Keratin biomaterials for treatment of ischemia

ABSTRACT

Provided herein are keratin compositions useful for treating ischemia and/or reperfusion injury, such as that associated with myocardial infarct, ischemic stroke, brain trauma such as traumatic brain injury, hypothermia, chronic wounds, and burns.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application Ser. No. 61/311,574, filed Mar. 8, 2010,the disclosure of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to keratin-based biomaterials and the usethereof for methods of treatment for ischemia and/or reperfusion injury.

BACKGROUND

Ischemia is an acute condition associated with an inadequate flow ofoxygenated blood to a part of the body, caused by the constriction orblockage of the blood vessels supplying it. This reduction in blood flowcan result from the blockage of a vessel by an embolus (blood clot), theblockage of a vessel due to atherosclerosis, etc. The restriction ofblood flow quickly causes necrosis in the effected tissues.

SUMMARY

Provided herein are keratin compositions and methods of treatingischemia and/or reperfusion injury (e.g., associated with myocardialinfarct, ischemic stroke, brain trauma such as traumatic brain injury,hypothermia, chronic wounds, and burns) in a subject in need thereof(e.g., a human subject) including administering the keratin compositionsto the subject in an amount effective to treat the ischemia and/orreperfusion injury.

In some embodiments, the keratin is keratose selected from the groupconsisting of: α-keratose, γ-keratose, α-kerateine, γ-keratein, keratinassociated proteins (KAP), and combinations thereof.

In some embodiments, the composition comprises (includes), consists ofor consists essentially of: (a) from 0.1 to 10 percent by weight ofkeratin; and (b) from 90 to 99.9 percent by weight of an electrolytesolution; wherein the keratose is solubilized in the electrolytesolution (e.g., normal saline) to form a homogeneous liquid compositionhaving (i) a pH of 7-8; (ii) an osmolarity of 200 to 500milliosmoles/Liter; and (iii) a viscosity of 2 to 20 centipoise, asdetermined at a temperature of 37 degrees Celsius in a Brookfieldviscometer having a cone and plate geometry with a cone angle of 0.02radians at a constant frequency of 30 rotations per minute.

In some embodiments, the compositions are administered in combinationwith a thrombolytic or an anticoagulant.

Compositions including keratin such as keratose and further including athrombolytic or anticoagulant are also provided.

Further provided is the use of a keratin composition as provided hereinfor the treatment of ischemia and/or reperfusion injury (e.g.,associated with myocardial infarct, ischemic stroke, brain trauma suchas traumatic brain injury, hypothermia, chronic wounds, and burns).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Dilation of a small diameter arteriole following top-loadadministration of KRF.

FIG. 2. Microvessel diameters measured in the cremaster muscle followingtop-load of fluid.

FIG. 3. Arteriole diameter before (left) and 20 minutes after (right)administration of 20% KAP-containing fluid in a rat.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Provided herein according to some embodiments are keratin fluids whichare useful to increase and/or restore blood flow and perfusion toischemic cells and tissues, and the use of these fluids to treatpatients in need of such therapy for conditions such as myocardialinfarct, stroke, traumatic brain injury (TBI) including mild TBI andother brain trauma, hypothermia, chronic wounds, burns, and otherconditions of ischemia.

The disclosures of all cited United States Patent references are herebyincorporated by reference to the extent they are consistent with thedisclosure herein. As used herein in the description of the inventionand the appended claims, the singular forms “a,” “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. Furthermore, the terms “about” and “approximately”as used herein when referring to a measurable value such as an amount ofa compound, dose, time, temperature, and the like, is meant to encompassvariations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specifiedamount. Also, as used herein, “and/or” refers to and encompasses any andall possible combinations of one or more of the associated listed items,as well as the lack of combinations when interpreted in the alternative(“or”).

According to some embodiments, the administration of keratincompositions as described herein increases blood vessel diameter (normaland/or vasoconstricted), along with blood flow, such that a blockage canbe overcome. The keratin composition of some embodiments causessignificant vasodilation independent of viscosity. In some embodiments,blood vessel diameter is increased by between 10, 15, 20 or 30% and 40,50, 60 or 70%. In other embodiments, blood vessel diameter is increasedby about 2%, about 5%, about 10%, about 15%, 20%, about 25%, about 30%,about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,about 100%, about 125%, about 150%, about 175%, about 200%, about 225%,about 250%, about 275%, about 300%, or more, than prior toadministration of a keratin composition.

“Ischemia” is a restriction, shortage or blockage in blood supply due toevents in the blood vessels such as vessel constriction, blockage bythrombosis or embolism, etc., often with resultant tissue damage and/ordysfunction. A “reperfusion injury” is an injury in which tissue isdamaged upon return of blood supply after a period of ischemia.

“Myocardial infarct” or “myocardial infarction,” commonly known as aheart attack, is an interruption in blood supply to part of the heartmuscle, causing myocardial cell death.

“Stroke” or “cerebrovascular accident” is an interruption in bloodsupply to part of the brain, often causing brain tissue damage and lossof brain function. “Ischemic stroke” or “cerebral infarction”, is causedby a blockage of blood vessels such as thrombosis, embolism, or systemichypoperfusion (e.g., shock).

Ischemia and/or reperfusion injury may also be a factor in traumaticbrain injury (TBI) including mild TBI and other brain trauma,hypothermia (a body temperature below 95 degrees Fahrenheit), chronicwounds, burns, etc. Thus, in some embodiments compositions taught hereinare useful in the treatment of these conditions.

In some embodiments, keratin compositions are administered incombination with an anticoagulant and/or a thrombolytic. Theadministration of two or more compounds “in combination” or “inconjunction” means that the two compounds are administered closelyenough in time to have an additive and/or synergistic effect. The twocompounds may be administered simultaneously (concurrently) orsequentially. Simultaneous administration may be carried out by mixingthe compounds prior to administration, or by administering the compoundsat the same point in time but at different anatomic sites or usingdifferent routes of administration.

Compositions comprising keratin and further comprising an anticoagulantare provided. Anticoagulants are pharmaceutical agents that decrease thegrowth of blood clots. Anticoagulants include heparin.

Compositions comprising keratin and further comprising a thrombolyticare provided. Thrombolytics are pharmaceutical agents that break down orreduce the size of blood clots, and include tissue plasminogen activator(tPA) and analogs thereof. Thrombolytics include streptokinase,urokinase, alteplase, reteplase, and tenecteplase.

To produce keratin biomaterials as described herein, sub-families ofkeratin proteins may be isolated, and in some embodiments recombinedinto a reconstituted composition. “Reconstituted composition” as usedherein means a composition comprising different ratios of independentlyisolated fractions of keratin materials, including, but not limited to,alpha-keratose, acidic alpha-keratose, basic alpha-keratose,gamma-keratose, acidic gamma-keratose, basic gamma-keratose,alpha-kerateine, acidic alpha-kerateine, basic alpha-kerateine,gamma-kerateine, acidic gamma-kerateine, basic gamma-kerateine, KAPs,alpha-keratose monomers, or alpha-kerateine monomers. The composition iscreated by mixing together the desired proportions of the isolatedfractions in solid, liquid, or hydrogel form. In some preferredembodiments, the reconstituted composition is substantially free ofKAPs. In other preferred embodiments, the reconstituted composition issubstantially free of alpha-keratose monomers and/or alpha kerateinemonomers.

In some embodiments, the composition includes from 0.01, 0.1, 0.5, 1, or2% to 3, 4, 5, 10, 25, 50 or 70% by weight of keratin. Thus, in someembodiments, compositions of the invention comprise about 0.1%, about0.25%, about 0.5%, about 0.75%, about 1%, about 1.25%, about 1.5%, about1.75%, about 2%, about 2.25%, about 2.5%, about 2.75%, about 3%, about3.25%, about 3.5%, about 3.75%, about 4%, about 4.25%, about 4.5%, about4.75%, about 5%, about 5.25%, about 5.5%, about 5.75%, about 6%, about6.25%, about 6.5%, about 6.75%, about 7%, about 7.25%, about 7.5%, about7.75%, about 8%, about 8.25%, about 8.5%, about 8.75%, about 9%, about9.25%, about 9.5%, about 9.75%, or about 10% by weight of a keratin suchas an alpha keratose, an alpha kerateine, keratin associate proteins(KAP), or a combination thereof. For example, compositions of theinvention may comprise 0.1%, 0.25%, 0.5%, 0.75%, 1%, 1.25%, 1.5%, 1.75%,2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%,5%, 5.25%, 5.5%, 5.75%, 6%, 6.25%, 6.5%, 6.75%, 7%, 7.25%, 7.5%, 7.75%,8%, 8.25%, 8.5%, 8.75%, 9%, 9.25%, 9.5%, 9.75%, or 10% by weight of akeratin such as an alpha keratose, an alpha kerateine, keratin associateproteins (KAP), or a combination thereof.

Also, in some embodiments, compositions of the invention comprise about0.1%, about 0.25%, about 0.5%, about 0.75%, about 1%, about 1.25%, about1.5%, about 1.75%, about 2%, about 2.25%, about 2.5%, about 2.75%, about3%, about 3.25%, about 3.5%, about 3.75%, about 4%, about 4.25%, about4.5%, about 4.75%, about 5%, about 5.25%, about 5.5%, about 5.75%, about6%, about 6.25%, about 6.5%, about 6.75%, about 7%, about 7.25%, about7.5%, about 7.75%, about 8%, about 8.25%, about 8.5%, about 8.75%, about9%, about 9.25%, about 9.5%, about 9.75%, or about 10% by weight analpha keratose, an alpha kerateine, a gamma keratose, a gamma kerateine,keratin associate proteins (KAP), or a combination thereof. In yet otherembodiments, compositions of the invention comprise 0.1%, 0.25%, 0.5%,0.75%, 1%, 1.25%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%,3.75%, 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75%, 6%, 6.25%, 6.5%,6.75%, 7%, 7.25%, 7.5%, 7.75%, 8%, 8.25%, 8.5%, 8.75%, 9%, 9.25%, 9.5%,9.75%, or about 10% by weight of an alpha keratose, an alpha kerateine,a gamma keratose, a gamma kerateine, keratin associate proteins (KAP),or a combination thereof.

In some embodiments, compositions of the invention include KAP. Inalternative embodiments, compositions of the invention are free orsubstantially free of KAP.

Keratins are a family of proteins found in the hair, skin, and othertissues of vertebrates. Hair is a unique source of human keratinsbecause it is one of the few human tissues that is readily available andinexpensive. Although other sources of keratins are acceptablefeedstocks for the present invention, (e.g., wool, fur, horns, hooves,beaks, feathers, scales, and the like), human hair is preferred for usewith human subjects because of its biocompatibility. The human hair canbe end-cut, as one would typically find in a barber shop or salon.

“Keratin” or “keratin derivative” as used herein refers to any keratinfractionation, derivative, subfamily, etc., or mixtures thereof, aloneor in combination with other keratin derivatives or other ingredients,including, but not limited to, alpha keratose, gamma keratose, alphakerateine, gamma kerateine, meta keratin, keratin intermediatefilaments, and combinations thereof, including the acidic and basicconstituents thereof unless specified otherwise, along with variationsthereof that will be apparent to persons skilled in the art in view ofthe present disclosure.

“Subjects” are generally human subjects and include, but are not limitedto, “patients.” The subjects may be male or female and may be of anyrace or ethnicity, including, but not limited to, Caucasian,African-American, African, Asian, Hispanic, Indian, etc. The subjectsmay be of any age, including newborn, neonate, infant, child,adolescent, adult, and geriatric.

Subjects also include animal subjects, particularly mammalian subjectssuch as canines, felines, bovines, caprines, equines, ovines, porcines,rodents (e.g., rats and mice), lagomorphs, non-human primates, etc.,for, e.g., veterinary medicine, laboratory research and/orpharmaceutical drug development purposes.

“Treat” refers to any type of treatment that imparts a benefit to apatient, e.g., a patient who is injured or who is afflicted with or atrisk for developing a disease (e.g., stroke, myocardial disease,cardiovascular disease, etc.). Treating includes actions taken andactions refrained from being taken for the purpose of improving thecondition of the patient (e.g., the relief of one or more symptoms),delay in the onset or progression of the disease, etc. Also, in someembodiments, treating comprises inhibiting, reducing, and/or preventingthe disease or symptoms thereof in a subject in need.

In some embodiments, compositions comprising, consisting of orconsisting essentially of keratin potentiates the release of strongvasodilators and induces net intravascular transport of water,increasing circulating volume. This results in peripheral vasodilationand increased cardiac contractility (the ability of the cardiac muscleto contract at a given fiber length), which translates into lowercardiac work and better tissue perfusion.

Vital signs which may be measured and/or monitored in connection withthe treatments described herein include, but are not limited to, meanarterial blood pressure (MAP—average arterial pressure during a cardiaccycle), shock index, base deficit, renal output, kidney function,hematocrit, blood gases, etc., which may be indicative of ischemiaand/or reperfusion injury. In some embodiments, the invention providesmethods of treating, inhibiting, reducing, and/or preventing the diseaseor at least one symptom thereof wherein a patient in need wherein saidpatient exhibits a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% improvement in thedisease or symptom thereof as compared to prior to administration of thecomposition to the patient.

In other embodiments, the invention provides methods of treating,inhibiting, reducing, and/or preventing the disease or at least onesymptom thereof wherein a subject in need wherein said patient exhibitsimprovement in about 1 min, about 2 min, about 5 min, about 10 min,about 15 min, about 30 min, about 60 min, about 120 min, or about 180min as compared to prior to administration of the composition to thesubject.

In other embodiments, the invention provides compositions that increasevasodilation over other fluids used for reperfusion and/or ischemiaapplications. Such fluids include, but are not limited, to Hetastarchand PBS. Thus, in some embodiments, compositions of the inventionincrease vasodilation about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about95%, about 100%, about 125%, about 150%, about 175%, about 200%, about250%, or about 300% or more than the vasodilation exhibited with otherfluids.

In some embodiments, subjects to be treated may have a systolic bloodpressure of between 120, 130, 140 or 150 and 180, 200, 250 or 300 mm Hg,and/or a diastolic blood pressure of between 80, 85, 90, 95, or 100 and120, 125 or 130 mm Hg (hypertension or prehypertension). In someembodiments, subjects to be treated may have a systolic blood pressureof between 80, 90, 100 or 110 and 120, 130 or 140 mm Hg, and/or adiastolic blood pressure of between 40, 50, or 60 and 70, 80 or 90 mm Hg(normal or low blood pressure).

In some embodiments, subjects to be treated may have a resting pulse offrom 60, 70, 80, 90, 100, 110 or 120 to 150, 16 or 170 beats per minute,which may indicate tachycardia depending on the subject (normal ratebeing from 60-100 beats per minute, taking into consideration that womentend to have higher resting pulses than men, and an athlete may have anormal resting pulse of 40 beats per minute). In some embodiments,subjects to be treated may have a resting pulse of from 10, 20, 30 or 40to 50, 60, 70 or 80 beats per minute, which may indicate normal or a lowpulse rate, again, depending of the subject in question.

In some embodiments, subjects to be treated may have a restingrespiration rate of from 20, 25, 30 or 35 to 40, 45 or 50 breaths perminute or more (the normal range being usually 15-20 breaths perminute). In some embodiments, subjects may have a resting respirationrate of 15 breaths per minute or less, down to zero breaths per minutein the case of a subject who has become unconscious.

Extracted keratin solutions are known to spontaneously self-assemble atthe micron scale (see, e.g., Thomas et al., Int J Biol Macromol 1986;8:258-64; van de Löcht, Melliand Textilberichte 1987; 10:780-6).Self-assembly results in a highly regular structure with reproduciblearchitectures, dimensionality, and porosity. When the keratin isprocessed correctly, this ability to self-assemble can be preserved andused to create regular architectures on a size scale conducive tomolecular infiltration and/or attachment. When keratins are hydrolyzed(e.g., with acids or bases), their molecular weight is reduced, and theylose the ability to self-assemble. Therefore, processing conditions thatminimize hydrolysis are preferred.

Soluble keratins can be extracted from human hair fibers by oxidation orreduction using methods known in the art (see, for example, Rouse J G,Van Dyke M E. A review of keratin-based biomaterials for biomedicalapplications. Materials 2010; 3:999-1014). These methods typicallyemploy a two-step process whereby the crosslinked structure of keratinsis broken down by either oxidation or reduction. In these reactions, thedisulfide bonds in cystine amino acid residues are cleaved, renderingthe keratins soluble. The cuticle is essentially unaffected by thistreatment, so the majority of the keratins remain trapped within thecuticle's protective structure. In order to extract these keratins, asecond step using a denaturing solution is employed. Alternatively, inthe case of reduction reactions, these steps can be combined. Denaturingsolutions known in the art include urea, transition metal hydroxides,surfactant solutions, and combinations thereof. Preferred methods useaqueous solutions of tris base(2-Amino-2-(hydroxymethyl)-1,3-propanediol) in concentrations between0.1 and 1.0 M, and urea solutions between 0.1 and 10M, for oxidation andreduction reactions, respectively.

If one employs an oxidative treatment, the resulting keratins arereferred to as “keratoses.” If a reductive treatment is used, theresulting keratins are referred to as “kerateines” (See Scheme 1).

Crude (unfractionated) extracts of keratins, regardless of redox state,can be further refined into matrix (KAP and gamma), alpha, and/orcharged (acidic or basic) fractions by a variety of methods such asisoelectric precipitation, dialysis, or high performance liquidchromatography (HPLC), as desired. In a crude extract, the alphafraction begins to precipitate below pH 6 and is essentially completelyprecipitated by pH 4.2.

In some embodiments, KAP co-precipitate with the alpha fraction, therebyproducing an alpha/KAP mixture. See Rogers et al., “Human HairKeratin-Associated Proteins (KAPs),” Int'l ref. cytol. 251:209-263(2006).

High molecular weight keratins, or “alpha keratins,” (alpha helical),are thought to originate from the microfibrillar regions of the hairfollicle, and monomers of alpha keratins typically range in molecularweight from about 40-85 kiloDaltons. They may also exist ashigher-ordered structures, i.e., complexed into multimeric forms witheach other or other keratins. Low molecular weight keratins, or “gammakeratins,” or keratin-associated proteins (globular), are thought tooriginate from the matrix regions of the hair follicle, and typicallyrange in molecular weight from about 3-30 kiloDaltons for KAP and 10-15kiloDaltons for gamma keratins (see Rouse J G, Van Dyke M E. A review ofkeratin-based biomaterials for biomedical applications. Materials 2010;3:999-1014).

In some embodiments, the keratin preparations (particularly alpha and/orgamma kerateine and alpha and/or gamma keratose) have an averagemolecular weight of from about 10, 20, 30, 40 or 50 to 70, 80, 85, 90,95 or 100 kiloDaltons. Other keratin derivatives, particularly complexedkeratins, may have higher average molecular weights, e.g., up to 200 or300 kiloDaltons.

Even though alpha and gamma keratins possess unique properties, theproperties of subfamilies of both alpha and gamma keratins can only berevealed through more sophisticated means of purification andseparation. Additional properties that are beneficial emerge and can beoptimized upon further separation and purification of crude keratinextracts. Many protein purification techniques are known in the art, andrange from the most simplistic, such as fractional precipitation, to themore complex, such as immunoaffinity chromatography. For extensivetreatment of this subject, see Scopes R K (editor) Protein Purification:Principles and Practice (3rd ed. Springer, New York 1993); Roe S,Protein Purification Techniques: A Practical Approach (2nd ed. OxfordUniversity Press, New York 2001); Hatti-Kaul R and Mattiasson B,Isomation and Purification of Proteins (Marcel Dekker AG, New York2003). For example, sub-families of acidic and basic keratin areseparable by moving boundary electrophoresis. A preferred method offractionation is ion exchange chromatography. It was discovered thatthese fractions possess unique properties, such as their differentialeffects on blood cell aggregation (see, e.g., U.S. Pat. No. 7,439,012 toVan Dyke).

In some embodiments, the keratin derivative comprises, consists orconsists essentially of a particular fraction or subfraction of keratin.The derivative in some embodiments may comprise, consist or consistessentially of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 99percent by weight of said fraction or subfraction (or more).

In some embodiments, the keratin derivative comprises, consists of, orconsists essentially of acidic and/or basic, alpha and/or gammakeratose, where the keratose comprises, consists of or consistsessentially of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 99percent by weight of acidic and/or basic, alpha and/or gamma keratose(or more).

In some embodiments, the composition comprises from 0.1 to 10 percent byweight of a keratin. In other embodiments, the composition comprisesfrom 90 to 99.9 percent by weight of an electrolyte solution; whereinthe keratin is solubilized in the electrolyte solution (e.g., normalsaline) to form a homogeneous liquid composition. In furtherembodiments, the homogenous liquid composition may have any of: (i) a pHof 6 or 7 to 8 or 9; (ii) an osmolarity of 200 to 500milliosmoles/Liter; and (iii) a viscosity of 2 to 20 centipoise. In yetfurther embodiments, the viscosity of said homogenous liquid compositionmay be determined at a temperature of 37 degrees Celsius in a Brookfieldviscometer. In still yet a further embodiment, the viscosity may exhibita cone and plate geometry with a cone angle of 0.02 radians at aconstant frequency of 30 rotations per minute.

Keratose Production.

A preferred method for the production of keratoses is by oxidation withhydrogen peroxide, peracetic acid, or performic acid. A most preferredoxidant is peracetic acid. Preferred concentrations range from 1 to 10weight/volume percent, the most preferred being approximately 2 w/v %.Those skilled in the art will recognize that slight modifications to theconcentration can be made to affect varying degrees of oxidation, withconcomitant alterations in reaction time, temperature, and liquid tosolid ratio. It has also been discussed by Crewther et al. thatperformic acid offers the advantage of minimal peptide bond cleavagecompared to peracetic acid. However, peracetic acid offers theadvantages of cost and availability. A preferred oxidation temperatureis between 0 and 100 degrees Celsius. A most preferred oxidationtemperature is 37° C. A preferred oxidation time is between 0.5 and 24hours. A most preferred oxidation time is 10 hours. A preferred liquidto solid ratio is from 5 to 100:1. A most preferred ratio is 20:1. Afteroxidation, the hair can be rinsed free of residual oxidant using acopious amounts of purified water.

The keratoses may be extracted from the oxidized hair using an aqueoussolution of a denaturing agent. Protein denaturants are well known inthe art, but preferred solutions include urea, transition metalhydroxides (e.g. sodium and potassium hydroxide), ammonium hydroxide,and tris(hydroxymethyl)aminomethane (Trizma® base). A preferred solutionis Trizma base in the concentration range from 0.01 to 1M. A mostpreferred concentration is 0.1M. Those skilled in the art will recognizethat slight modifications to the concentration can be made to effectvarying degrees of extraction, with concomitant alterations in reactiontime, temperature, and liquid to solid ratio. A preferred extractiontemperature is between 0 and 100 degrees Celsius. A most preferredextraction temperature is 37° C. A preferred extraction time is between0.5 and 24 hours. A most preferred extraction time is 2 hours. Apreferred liquid to solid ratio is from 5 to 100:1. A most preferredratio is 40:1. Additional yield can be achieved with subsequentextractions with dilute solutions of Trizma base or purified water.After extraction, the residual solids can be removed from solution bycentrifugation and/or filtration.

Residual denaturing agent may be removed by dialysis against purifiedwater or buffer solution. Concentration of the dialysis retentate may befollowed by lyophilization or spray drying, resulting in a dry powdermixture of gamma and alpha keratoses as well as KAP. Alternately, analpha/KAP mixture may be isolated from the crude extract solution bydropwise addition of acid until the pH of the solution reachesapproximately 4.2. Preferred acids include sulfuric, hydrochloric, andacetic. A most preferred acid is concentrated hydrochloric acid.Precipitation of the alpha/KAP fraction begins at around pH 6.0 andcontinues until approximately 4.2. Fractional precipitation can beutilized to isolate different ranges of protein with differentisoelectric properties. Precipitated alpha/KAP can be recovered bycentrifugation, filtration, or the like. The alpha/KAP mixture isfurther purified by re-dissolving the solids in a denaturing solution.The same denaturing solutions as those utilized for extraction can beused. However, a preferred denaturing solution is Trizma base. Ethylenediamine tetraacetic acid (EDTA) can be added to complex and remove tracemetals found in hair. A preferred denaturing solution is 100 mM trisbase with 20 mM EDTA or DI water with 20 mM EDTA, if desired. If thepresence of trace metals is not detrimental to the intended application,the EDTA step may be omitted. The alpha/KAP mixture can bere-precipitated from this solution by dropwise addition of hydrochloricacid to a final pH of 4.2. Isolation of the solid may be done bycentrifugation, filtration or the like. This process can be repeatedseveral times to further purify the alpha/KAP mixture, if desired,although significant destruction of amide bonds should be avoidedaccording to some embodiments. In another preferred embodiment, thealpha/KAP fraction can be isolated from gamma-keratose by dialysis.Providing a high nominal low molecular weight cutoff membrane such thatthe gamma passes through the membrane and the alpha/KAP is retained caneffect such separation. Preferred membranes are those having nominal lowmolecular weight cutoffs of 15,000 to 100,000 Da. Most preferredmembranes are those having nominal low molecular weight cutoffs of30,000 and 100,000 Da.

The gamma keratose fraction can be isolated by addition to awater-miscible non-solvent. Suitable non-solvents include ethanol,methanol, acetone, and the like. A most preferred non-solvent isethanol. To effect precipitation, the gamma keratose solution can beconcentrated by removal of excess water. This can be done using vacuumdistillation, falling film evaporation, microfiltration, etc. Afterconcentration, the gamma keratose solution is added dropwise to anexcess of cold non-solvent. A most preferred method is to concentratethe gamma keratose solution to approximately 10 weight/volume (w/v) %protein and add it dropwise to an 8-fold excess of cold ethanol. Theprecipitated gamma keratose can be isolated by centrifugation orfiltration and dried. Suitable methods for drying include freeze drying(lyophilization), air drying, vacuum drying, or spray drying. A mostpreferred method is freeze drying. Alternately, the gamma keratose canbe isolated by dialysis against purified water or buffer solution.Preferred membranes for dialysis are those having nominal low molecularweight cutoffs between 1,000 and 5,000 Da. Most preferred membranes fordialysis are those having nominal low molecular weight cutoffs of 3,000and 5,000 Da. This solution can be concentrated by additional dialysisand reduced to a dry powder by lyophilization or spray drying.

Several different approaches to further purification can be employed tokeratose solutions (e.g., crude, alpha or gamma keratose). Care must betaken, however, to choose techniques that lend themselves to keratin'sunique solubility characteristics. One of the most simple separationtechnologies is isoelectric precipitation. Another general method forseparating keratins is by chromatography. Several types ofchromatography can be employed to fractionate keratin solutionsincluding size exclusion or gel filtration chromatography, affinitychromatography, isoelectric focusing, gel electrophoresis, ion exchangechromatography, and immunoaffinity chromatography. These techniques arewell known in the art and are capable of separating compounds, includingproteins, by the characteristics of molecular weight, chemicalfunctionality, isoelectric point, charge, or interactions with specificantibodies, and can be used alone or in any combination to affect highdegrees of separation and resulting purity.

A preferred purification method is ion exchange (IEx) chromatography.IEx chromatography is particularly suited to protein separation owningto the amphiphilic nature of proteins in general and keratins inparticular. Depending on the starting pH of the solution, and thedesired fraction slated for retention, either cationic or anionic IEx(CIEx or AIEx, respectively) techniques can be used. For example, at apH of 7 and above, both gamma and alpha/KAP keratose fractions aresoluble and above their isoelectric points. As such, they are anionicand can be bound to an anionic exchange resin. However, if the pH isbelow approximately 6, the alpha in the alpha/KAP fraction will not bindto the resin and instead passes through a column packed with such resin.A preferred solution for AIEx chromatography is alpha/KAP solution,isolated as described previously, in weak buffer solution at aconcentration between 0 and 5 weight/volume %. A preferred concentrationis approximately 2 w/v %. It is preferred to keep the ionic strength ofsaid solution initially quite low to facilitate binding to the AIExcolumn. This is achieved by using a minimal amount of acid to titrate apurified water solution of the keratin to between pH 5.3 and 6. A mostpreferred pH is 5.3. This solution can be loaded onto an AIEx columnsuch as DEAE-Sepharose or Q-Sepharose, or processed in bulk without theuse of a column. The solution that passes through the column can becollected and further processed as described previously to isolate afraction of alpha powder.

The basic fraction (including KAP) binds readily due to its lowerisoelectric point, and can be washed off the column using saltingtechniques known in the art. A preferred elution medium is sodiumchloride solution. A preferred concentration of sodium chloride isbetween 0.1 and 2M. A most preferred concentration is 2M. The pH of thesolution is preferred to be between 6 and 12. A most preferred pH is 11.In order to maintain stable pH during the elution process, a buffer saltcan be added. A preferred buffer salt is Trizma base. A preferredconcentration of Trizma base is 100 mM. Those skilled in the art willrecognize that slight modifications to the salt concentration and pH canbe made to affect the elution of keratin fractions with differingproperties. It is also possible to use different salt concentrations andpH's in sequence, or employ the use of salt and/or pH gradients toproduce different fractions. Regardless of the approach taken, however,the column eluent can be collected and further processed as describedpreviously to isolate purified fractions of alpha-keratose powders.

A complimentary procedure is also feasible using CIEx techniques.Namely, the alpha/KAP solution can be added to a cation exchange resinsuch as SP Sepharose (strongly cationic) or CM Sepharose (weaklycationic), and the basic (KAP) fraction collected with the pass through.The retained alpha fraction can be isolated by salting as previouslydescribed.

Kerateine Production.

Similar to the methods described above for extraction and purificationof keratoses, kerateines can be produced by reduction of hair fiberswith thioglycolic acid or beta-mercaptoethanol. A most preferredreductant is thioglycolic acid (TGA). Preferred concentrations rangefrom 0.1 to 10M, the most preferred being approximately 1.0M or 0.5M.Those skilled in the art will recognize that slight modifications to theconcentration can be made to effect varying degrees of reduction, withconcomitant alterations in pH, reaction time, temperature, and liquid tosolid ratio. A preferred pH is between 9 and 11. A most preferred pH is10.2. The pH of the reduction solution is altered by addition of base.Preferred bases include transition metal hydroxides and ammoniumhydroxide. A most preferred base is sodium hydroxide. The pH adjustmentis affected by dropwise addition of a saturated solution of sodiumhydroxide in water to the reductant solution. A preferred reductiontemperature is between 0 and 100 degrees Celsius. A most preferredreduction temperature is 37° C. A preferred reduction time is between0.5 and 24 hours. A most preferred reduction time is 12 hours. Apreferred liquid to solid ratio is from 5 to 100:1. A most preferredratio is 20:1. Unlike the previously described oxidation reaction,reduction is carried out at basic pH. That being the case, keratins arehighly soluble in the reduction media and are expected to be extracted.The reduction solution may therefore be combined with the subsequentextraction solutions and processed accordingly.

Reduced keratins are not as hydrophilic as their oxidized counterparts.As such, reduced hair fibers will not swell and split open as willoxidized hair, resulting in relatively lower yields. Another factoraffecting the kinetics of the reduction/extraction process is therelative solubility of kerateines. The relative solubility rankings inwater, from most to least soluble, isgamma-keratose>alpha-keratose>gamma-kerateine>alpha-kerateine.Consequently, extraction yields from reduced hair fibers are not ashigh. This being the case, subsequent extractions are conducted withadditional reductant plus denaturing agent solutions. Typical solutionsfor subsequent extractions include TGA plus urea, TGA plus Trizma base,or TGA plus sodium hydroxide. After extraction, crude fractions ofalpha/KAP and gamma kerateine can be isolated using the proceduresdescribed for keratoses. However, precipitates of gamma and alpha/KAPkerateine re-form their cystine crosslinks upon exposure to oxygen.Precipitates should, therefore, preferably be re-dissolved quickly so asto avoid insolubility during the purification stages, or precipitated inthe absence of oxygen.

Purification of kerateine solutions can be conducted similar to thosedescribed for keratoses. Those skilled in the art will recognize thatthe chemical nature of kerateines varies from that of keratoses,primarily in the fate of pendant sulfur groups that will alter chemicalproperties such as isoelectric points. As such, modifications in theconditions for separation techniques such as ion exchange chromatographyare needed for optimization.

In some embodiments, the keratin derivative comprises, consists orconsists essentially of a particular fraction or subfraction of keratin.The derivative in some embodiments may comprise, consist or consistessentially of at least 80, 90, 95 or 99 percent by weight of saidfraction or subfraction (or more).

In some embodiments, the keratin derivative comprises, consists of, orconsists essentially of acidic and/or basic, alpha and/or gammakeratose, where the keratose comprises, consists of or consistsessentially of at least 80, 90, 95 or 99 percent by weight of acidicand/or basic, alpha and/or gamma keratose (or more).

In some embodiments, the keratin derivative comprises, consists of, orconsists essentially of acidic and/or basic, alpha and/or gammakeratose, where the keratose comprises, consists of, or consistsessentially of at least 80, 90, 95 or 99 percent by weight of acidicand/or basic, alpha and/or gamma keratose (or more). In otherembodiments, the keratin derivative comprises, consists of, or consistsessentially of alpha/KAP keratose, where the keratose comprises, consistof, or consists essentially of at least 80, 90, 95 or 99 percent byweight of alpha/KAP keratose (or more).

In some embodiments, the keratin derivative comprises, consists of, orconsists essentially of acidic and/or basic, alpha and/or gammakerateine, where the kerateine comprises, consists of or consistsessentially of at least 80, 90, 95 or 99 percent by weight of acidicand/or basic, alpha and/or gamma kerateine (or more). In otherembodiments, the keratin derivative comprises, consists of, or consistsessentially of alpha/KAP kerateine, where the kerateine comprises,consist of, or consists essentially of at least 80, 90, 95 or 99 percentby weight of alpha/KAP keratose (or more).

The basic alpha keratose is preferably produced by separating basicalpha keratose from a mixture comprising acidic and basic alphakeratose, e.g., by ion exchange chromatography, and optionally the basicalpha keratose has an average molecular weight of from 10 to 100 or 200kiloDaltons. More preferably, the average molecular weight is from 30 or40 to 90 or 100 kiloDaltons. Optionally, but in some embodimentspreferably, the process further comprises the steps of re-dissolvingsaid basic alpha-keratose in a denaturing and/or buffering solution,optionally in the presence of a chelating agent to complex trace metals,and then re-precipitating the basic alpha keratose from the denaturingsolution. It will be appreciated that the composition preferablycontains not more than 5, 2, 1, or 0.1 percent by weight of acidic alphakeratose, or less.

The acidic alpha keratose may be produced by a reciprocal of theforegoing technique: that is, by separating and retaining acidic alphakeratose from a mixture of acidic and basic alpha keratose, e.g., by ionexchange chromatography, and optionally the acidic alpha keratose has anaverage molecular weight of from 10 to 100 or 200 kiloDaltons. Morepreferably, the average molecular weight is from 30 or 40 to 90 or 100kiloDaltons. Optionally, but in some embodiments preferably, the processfurther comprises the steps of re-dissolving said acidic alpha-keratosein a denaturing solution and/or buffering solution, optionally in thepresence of a chelating agent to complex trace metals, and thenre-precipitating the basic alpha keratose from the denaturing solution.It will be appreciated that the composition preferably contains not morethan 5, 2, 1, or 0.1 percent by weight of basic alpha keratose, or less.

Basic and acidic fractions of other keratoses (e.g., KAP and gammakeratose) can be prepared in like manner as described above for basicand acidic alpha keratose.

Basic alpha kerateine is preferably produced by separating basic alphakerateine from a mixture of acidic and basic alpha kerateine, e.g., byion exchange chromatography, and optionally the basic alpha kerateinehas an average molecular weight of from 10 to 100 or 200 kiloDaltons.More preferably, the average molecular weight is from 30 or 40 to 90 or100 kiloDaltons. Optionally, but preferably, the process furtherincludes the steps of re-dissolving said basic alpha-kerateine in adenaturing and/or buffering solution, optionally in the presence of achelating agent to complex trace metals, and then re-precipitating thebasic alpha kerateine from the denaturing solution. It will beappreciated by those of skill in the art that the composition preferablycontains not more than 5, 2, 1, or 0.1 percent by weight of acidic alphakerateine, or less.

The acidic alpha kerateine may be produced by a reciprocal of theforegoing technique; that is, by separating and retaining acidic alphakerateine from a mixture of acidic and basic alpha kerateine, e.g., byion exchange chromatography, and optionally the acidic alpha kerateinehas an average molecular weight of from 5 or 10 to 100 or 200kiloDaltons. Optionally, but preferably, the process further comprisesthe steps of re-dissolving said acidic alpha-kerateine in a denaturingand/or buffering solution), optionally in the presence of a chelatingagent to complex trace metals, and then re-precipitating the basic alphakerateine from the denaturing solution. It will be appreciated that thecomposition preferably contains not more than 5, 2, 1, or 0.1 percent byweight of basic alpha kerateine, or less.

Basic and acidic fractions of other kerateines (e.g., KAP and gammakerateine) can be prepared in like manner as described above for basicand acidic alpha kerateine. Gamma keratins are typically precipitated ina non-solvent such as ethanol.

As used herein, “acidic” keratins are those keratins that are protonatedat a predetermined pH such that they carry a net positive charge;“basic” keratins are those keratins that are de-protonated at apredetermined pH such that they carry a net negative charge. The keratinassociated proteins (KAP) as used herein carry a negative charge at thepredetermined pH and bind to an anionic exchange resin, and thus in someembodiments is included in the basic keratin fractions taught herein. Insome embodiments, the predetermined pH is between 5 and 7. In someembodiments, the pH is 6. For example, in some embodiments, keratose orkerateine is separated into acidic and basic fractions (e.g., by ionexchange chromatography) performed at a solution pH of 6, with theresulting acidic fraction including those keratins having a net positivecharge at pH 6, and the basic fraction including those keratins having anet negative charge at pH 6. Likewise, for separation at a predeterminedpH of 5.3, the acidic fraction will include those keratins having a netpositive charge at pH 5.3 and the basic fraction will include thosekeratins having a net negative charge at pH 5.3.

Those skilled in the art will recognize that the predetermined pH isselected to effect the best separation between acidic and basic proteinsbased upon their isoelectric points (see, e.g., Table 1), thoughsolubility at that pH should also be considered. When the pH of thesolution is between the isoelectric point of these acidic and basickeratin fractions, basic keratin proteins will be de-protonated to havea net negative charge and bind to an anionic media (e.g., DEAE-Sepharoseor Q-Sepharose (anion exchange)), while the acidic proteins will beprotonated to have a net positive charge and pass through the column,thereby effecting separation.

Residual reductant and denaturing agents can be removed from solution bydialysis. Typical dialysis conditions are 1 to 2% solution of kerateinesdialyzed against purified water. Those skilled in the art will recognizethat other methods exist for the removal of low molecular weightcontaminants in addition to dialysis (e.g. microfiltration,chromatography, and the like). The use of Trizma base is only requiredfor initial solubilization of the kerateines. Once dissolved, thekerateines are stable in solution without the denaturing agent forfinite periods. Therefore, the denaturing agent can be removed withoutthe resultant precipitation of kerateines. Regardless of thefractionation/purification process, the resulting kerateines can beconcentrated and lyophilized, similar to keratoses.

The higher the percentage of alpha keratose or alpha kerateine in thecomposition leads to decreased hydrolytic susceptibility. Conversely,lowering the percentage of alpha keratose or alpha kerateine in thecomposition leads to increased hydrolytic susceptibility.

Thus, in some embodiments, reconstituted compositions of the inventioncomprise about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1%,about 1.25%, about 1.5%, about 1.75%, about 2%, about 2.25%, about 2.5%,about 2.75%, about 3%, about 3.25%, about 3.5%, about 3.75%, about 4%,about 4.25%, about 4.5%, about 4.75%, about 5%, about 5.25%, about 5.5%,about 5.75%, about 6%, about 6.25%, about 6.5%, about 6.75%, about 7%,about 7.25%, about 7.5%, about 7.75%, about 8%, about 8.25%, about 8.5%,about 8.75%, about 9%, about 9.25%, about 9.5%, about 9.75%, or about10% by weight alpha keratose or alpha kerateine. In yet otherembodiments, reconstituted compositions of the invention comprise 0.1%,0.25%, 0.5%, 0.75%, 1%, 1.25%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%,3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75%, 6%,6.25%, 6.5%, 6.75%, 7%, 7.25%, 7.5%, 7.75%, 8%, 8.25%, 8.5%, 8.75%, 9%,9.25%, 9.5%, 9.75%, or 10% by weight alpha keratose or alpha kerateine.

Also, in some embodiments, reconstituted compositions of the inventioncomprise about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1%,about 1.25%, about 1.5%, about 1.75%, about 2%, about 2.25%, about 2.5%,about 2.75%, about 3%, about 3.25%, about 3.5%, about 3.75%, about 4%,about 4.25%, about 4.5%, about 4.75%, about 5%, about 5.25%, about 5.5%,about 5.75%, about 6%, about 6.25%, about 6.5%, about 6.75%, about 7%,about 7.25%, about 7.5%, about 7.75%, about 8%, about 8.25%, about 8.5%,about 8.75%, about 9%, about 9.25%, about 9.5%, about 9.75%, or about10% by weight gamma keratose or gamma kerateine. In yet otherembodiments, reconstituted compositions of the invention comprise 0.1%,0.25%, 0.5%, 0.75%, 1%, 1.25%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%,3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75%, 6%,6.25%, 6.5%, 6.75%, 7%, 7.25%, 7.5%, 7.75%, 8%, 8.25%, 8.5%, 8.75%, 9%,9.25%, 9.5%, 9.75%, or about 10% by weight gamma keratose or gammakerateine.

Further discussion of keratin preparations are found in U.S. PatentApplication Publication 2009/0004242 (Van Dyke), which is incorporatedby reference herein.

These sub-fractions of keratin have demonstrated interestingcharacteristics such as an ability to rapidly cause dilation of bloodvessels, including collapsed blood vessels, and either increasecirculation or restore it, respectively. Using the different fractionsof keratoses as described above, either alone or in combination, thefluid properties and therapeutic effects of the keratose solution can becontrolled. Unique features of this system include:

-   -   An ability to re-combine keratin fractions into reconstituted        keratins that have controllable physical properties    -   An ability to tailor physical and bio-compatibility properties        to the cardiovascular system    -   An ability to cause dilation of blood vessels upon intravenous        administration of a keratose fluid    -   An ability to restore and/or improve blood flow and tissue        perfusion with a keratose fluid    -   An ability to treat patients with ischemia-related injuries

The vasodilation effect of some keratin fractions coupled with thematerial properties of the keratin fractions are useful for ischemiareperfusion treatment. The keratin fractions can be modified by varyingthe percent concentrations of the various fractions and/or by changingextraction processes (i.e. using a 30 kDa MWCO membrane versus a 100 kDaMWCO membrane and/or by column separating KAP from alpha fractions).

Keratose fluid formation is accomplished simply by rehydrating sterilekeratose powder with saline or other isotonic solution (e.g. Ringer'slactate) under aseptic conditions. Different sub-fractions of keratosecan be used to achieve the desired characteristics of physicalproperties, bio- and blood-compatibility, and vasodilatory effect. Theproduct can take the form of a ready-to-inject sterile fluid, or toincrease shelf life, sterile, lyophilized keratose powder packaged in acontainer to which isotonic solution can be added. Alternatively, saltsto achieve osmotic and pH balance can be added to the powdered keratoseand water added. The added liquid can be sterile and added aseptically,or a sterile filtering apparatus can be added to the keratose powderpackaging such that the liquid is sterile filtered as it is being added.

Formulations.

Dry powders may be formed of keratin preparations described above inaccordance with known techniques such as freeze drying (lyophilization).In some embodiments, hydrogel compositions of the invention may beproduced by mixing such a dry powder composition form with an aqueoussolution to produce a composition having an electrolyte solution with akeratin solubilized therein. The mixing step can be carried out at anysuitable temperature, typically room temperature, and can be carried outby any suitable technique such as stirring, shaking, agitation, etc. Thesalts and other constituent ingredients of the electrolyte solution(e.g., all ingredients except the keratin derivative and the water) maybe contained entirely in the dry powder, entirely within the aqueouscomposition, or may be distributed between the dry powder and theaqueous composition. For example, in some embodiments, at least aportion of the constituents of the electrolyte solution is contained inthe dry powder.

In some embodiments, the compositions are sterile. In some embodiments,keratin solutions are sterile filtered and processed aseptically, orterminally sterilized using ethylene oxide, e-beam, gamma, or other lowtemperature method (i.e. <50° C.).

The keratin composition may be provided as a kit of sterile dry powderin one container and sterile aqueous solution in a separate containerfor mixing just prior to use. The composition preferably has a shelflife of at least 4 or 6 months (up to 2 or 3 years or more) at roomtemperature, prior to substantial loss of viscosity (e.g., more than 10or 20 percent) and/or structural integrity of the keratin gel orhydrogel.

The composition may be provided in a precursor solution asepticallypackaged in a suitable container. For example, a gel precursor solutioncan be provided in a glass ampule ready to use directly or afterdilution by the user. In the case of kerateine compositions, which canre-crosslink in the presence of oxygen in air, a sterile precursorsolution in a sealed ampule under an inert atmosphere (e.g. nitrogen)can be provided. A user would simply break open the ampule, mix in acompound of interest and use the solution directly or after dilution forproducing the gel containing the compounds of interest dispersedtherein.

In some embodiments, keratin biomaterial compositions can be formulatedfor a injection or as a surface treatment (e.g., for burn wounds).Formulations of the invention include those for parenteraladministration (e.g., subcutaneous, intramuscular, intradermal,intravenous, intra-arterial, intraperitoneal injection) or implantation.In one embodiment, administration is carried out intravascularly, eitherby simple injection, or by injection through a catheter positioned in asuitable blood vessel, such as a renal artery.

In some embodiments, compounds of interest are administered in atherapeutically effective amount. The therapeutically effective dosagecan be determined in accordance with procedures known to those skilledin the art.

Embodiments of the present invention are further detailed in thefollowing non-limiting examples.

EXAMPLES Example 1

Keratose as a hyperviscous hyperosmotic compound potentiates the releaseof strong vasodilators and induces net intravascular transport of water,increasing circulating volume. This results in peripheral vasodilationand increased cardiac contractility which translates into lower cardiacwork and better tissue perfusion. The null hypothesis was tested that aKeratose resuscitation fluid (KRF) would not induce more arteriolarvasodilation than a current plasma expander, Hetastarch (HS).

Methods:

Keratose Resuscitation Fluid (KRF) was prepared as a mixture ofacidic+basic alpha keratose that had been dialyzed against a 30K nominallow molecular weight cutoff membrane. It was provided at about 5 wt. %in normal saline, pH 7.4. A defined topload volume of resuscitationfluid (2.25 ml/100 g) was infused into euvolemic (normal blood volume)rats. Eleven rats received Keratose Resuscitation Fluid (KRF; matched towhole blood viscosity; ca. 5 wt. %), eleven rats received Hetastarch 6%in 0.9% Sodium Chloride Solution (Hextend) and a control group of sevenrats received Phosphate Buffered Saline (PBS). A cremaster musclemicrovascular preparation was used to measure changes in diameter ofarterioles 20 μm to 65 μm in size. Diameters were measured beforeinfusion and at five-minute intervals up to thirty minutes afterinfusion.

Results:

Analysis of variance showed significant differences (p<0.05) between thethree treatment groups at all time-points (FIG. 2). Bonferroni'smultiple comparison tests showed that KRF induced greater vasodilationthan PBS or Hetastarch. Hetastarch vasodilatilatory effects were notstatistically different than those obtained with PBS.

Discussion:

Toploaded KRF induced significant vasodilation in the cremastermicrovasculature (FIG. 1) compared to equivalent volumes of Hetastarchor PBS. KRF induced significant vasodilation in muscle microvasculaturecompared to HS or PBS.

Example 2

Crude keratose solution was further processed to separate it into alphaand KAP fractions. This was accomplished by removing the keratose(alpha/KAP) from dialysis (100K Da NLMWCO), titrating the solution to pH6.0, and loading the sample onto a glass column containing Q Sepharoseanion exchange resin. The resin was used according to the manufacturer'sinstructions and was conditioned with three volumes of 10 mM tris at pH6.0. After loading, the sample, the column was rinsed with an additionalthree volumes of 10 mM tris buffer. The flow through and rinsesolutions, representing the alpha fraction, were collected, dialyzed at30 KDa NLMWCO, and processed to a dry powder. The sample bound to theresin, representing the KAP fraction was washed off with three volumesof 100 mM tris at pH 8.0+2M sodium chloride. This solution was dialyzedat 3 KDa NLMWCO and further processed to a dry powder as previouslydescribed. A KAP-containing fluid was prepared by dissolving 20% sterileprotein in sterile phosphate buffered saline.

The animal protocol was approved by the Wake Forest University School ofMedicine institutional animal care and use committee. MaleSprague-Dawley rats (S-D)(Charles River Laboratories International Inc,Wilmington, Mass.) weighting 95-157 g were studied. All rats were housedin a room with a controlled temperature (20 to 22° C.) and a twelve-hourlight-dark cycle, with tap water and rodent chow provided ad libitum.The rats were anesthetized with urethane diluted in 0.9% sodium chloridesolution and injected intraperitoneally at a dose of 1 gram per kilogramof body weight or until adequate anesthesia was achieved. Subjects werepositioned on the surgery table and the proximal trachea was isolated. Asmall incision was made between tracheal cartilage rings and apolyethylene tube (PE205) was inserted into the trachea to ensuresufficient and adequate airflow throughout the experiment. The incisionwas then extended laterally towards the right clavicle until the jugularvein was isolated. A saline-filled catheter was inserted into thejugular vein and secured for later infusion of treatments into thevenous bloodstream.

The subjects were repositioned and an incision was made through thelateral aspect of the scrotum for cremaster muscle isolation. The fasciaoverlying the cremaster muscle was resected and the muscle incisedthrough the midline until its inner contents were exposed. The spermaticchord and its components were ligated and resected. The muscle wascompletely isolated from other tissues and spread over a glass pedestal,secured with 6-0 silk sutures and a glass cover slip was placed over itfor efficient transillumination and to maintain physiologic tissue PO₂.

Rats were transferred to a compound microscope (Olympus BH W1, Japan)equipped for videomicroscopy with fiber optic illumination (Fiber-LiteModel 190, Dolan-Jenner Industries, Inc, Woburn, Mass.). Under 40×objective (Nikon, Japan; n.a.=0.5), A2 and A3 arterioles with clearlydefined walls, 20 μm to 65 μm in diameter, were selected for study.Microvascular dimensions were measured from video images (MTI CCD-72T,Michigan City, Ind.).

Subjects were allowed to stabilize for one hour after preparation forthe vasculature to recover from any transient changes associated withthe surgical preparation. A toploadmodel was employed using a definedvolume of fluid (2.25 ml/100 g), which was infused intravenously to therats in a euvolemic state. This volume represented an increase in bloodvolume of approximately 33%. The fluids were warmed to body temperatureprior to injection and infused through the jugular catheter over aperiod of two minutes. Arteriolar diameter was measured prior toinfusion and at five-minute intervals up to thirty minutes afterinfusion. As shown in FIG. 3, the vessel diameter increased from 31.3 μmto 42 μm (about 34%)

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1.-10. (canceled)
 11. A composition comprising a keratin and furthercomprising a thrombolytic.
 12. The composition of claim 11, wherein saidkeratin is selected from the group consisting of: α-keratose,γ-keratose, keratin associated proteins (KAP), and combinations thereof.13. The composition of claim 11, wherein said keratin is a combinationof α-keratose and keratin associated proteins (KAP).
 14. The compositionof claim 13, wherein said α-keratose has an average molecular weight ofat least 30 kiloDaltons.
 15. The composition of claim 11, wherein saidcomposition comprises from 1 to 10% by weight of said keratin.
 16. Thecomposition of claim 11, wherein said keratin is from 3 to 5 percent byweight.
 17. The composition of claim 11, wherein said composition isformulated for parenteral administration.
 18. The composition of claim11, wherein said thrombolytic is selected from the group consisting of:streptokinase, urokinase, alteplase, reteplase, and tenecteplase.
 19. Acomposition comprising a keratin and further comprising ananticoagulant.
 20. The composition of claim 19, wherein said keratin isselected from the group consisting of: α-keratose, γ-keratose, keratinassociated proteins (KAP), and combinations thereof.
 21. The compositionof claim 19, wherein said keratin is a combination of α-keratose andkeratin associated proteins (KAP).
 22. The composition of claim 20,wherein said α-keratose has an average molecular weight of at least 30kiloDaltons.
 23. The composition of claim 19, wherein said compositioncomprises from 1 to 10% by weight of said keratin.
 24. The compositionof claim 19, wherein said keratin is from 3 to 5 percent by weight. 25.The composition of claim 19, wherein said composition is formulated forparenteral administration.
 26. The composition of claim 19, wherein saidanticoagulant comprises heparin.